1. Introduction

Two methods are available for the generation of transgenic C. elegans: microinjection and microparticle bombardment (see Transformation and microinjection). Until quite recently, the only one-step method for producing integrated transgenes, as opposed to extrachromasomal arrays,
has been the microparticle bombardment method. This method has long been used to transform plant tissues and mammalian tissue
slices, but was first published for use in C. elegans in 2001 (Lo et al., 1994; Lorence and Verpoorte, 2004; Praitis et al., 2001). Since its introduction the bombardment technique has grown in popularity and is widely used to generate low copy-number
transgenes integrated randomly within the C. elegans genome. Often called biolistic transformation, the technique involves accelerating micron sized gold particles coated with
DNA (bullets) toward a living target, essentially a “gene gun”. Most modern biolistic devices (e.g., Biorad PDS-1000/He Hepta)
use high-pressure helium gas to accelerate the particles within a vacuum chamber, targeting the biological specimens to be
transformed, in this case a large population of adult C. elegans hermaphrodites (Fitzpatrick-McElligott, 1992; Praitis, 2006). The gold particles become lodged randomly within the cells of the target animals. After several generations, heritably
transformed animals can be recovered, presumably due to the small fraction of DNA coated gold particles that lodge within
the germ cells of the parent animals.

This technique allows for the recovery of both low copy-number, randomly integrated, transgenes, as well as high copy-number
extrachromasomal arrays (Praitis, 2006; Praitis et al., 2001). The bombardment technique is not efficient on a per-animal basis and therefore depends upon genetic selection, usually
accomplished by including a wild-type copy of the unc-119 gene with the DNA of interest, and targeting a population of unc-119 mutant animals. unc-119 mutants grow slowly, are mostly paralyzed, and fail to form dauer larvae under starvation conditions. Selection for movement
and formation of dauers after starvation is very efficient and allows for facile recovery of transformed animals. The C. briggsae version of the unc-119 gene (2.2 kb) is smaller than the C. elegans version (5.5 kb) due to smaller introns, but rescues the C. elegansunc-119 mutant extremely well (Maduro and Pilgrim, 1996). Therefore the C. briggsae version of the unc-119 gene, rather than the C. elegansunc-119 gene, is found in some C. elegans expression vectors. Recent publications also report successful recovery of transgenic C. elegans lines after bombardment using drug resistance markers (Semple et al., 2012).

Low copy-number transgenes obtained by microparticle bombardment offer several advantages that make them experimentally useful.
The first advantage is that at least some of the low-copy lines obtained by this method allow stable expression of the transgenes
in the C. elegans germline (Transgenic solutions for the germline; Praitis, 2006; Praitis et al., 2001). Transgenes are prone to silencing in C. elegans germ cells. In fact simple extrachromasomal arrays are almost universally silent in the germ line (Kelly et al., 1997). For somatic cell expression, the low-copy transgenes produced by bombardment generally offer relatively even expression
among equivalent animals in a population and among equivalent cells within a single animal (Praitis et al., 2001). Other methods that can also improve germline expression of transgenes and the reproducibility of somatic cell expression
include complex extrachromosomal arrays, and single copy integrants (Mosci method) (Frokjaer-Jensen et al., 2012; Frokjaer-Jensen et al., 2008; Kelly et al., 1997; Zeiser et al., 2011).

4. Protocol

Here we describe a version of the microparticle bombardment protocol that we have developed over the last 10 years, derived
in large part by combining aspects of existing protocols obtained from the Geraldine Seydoux, Shai Shaham, and Ronald Plasterk labs. This variation of the bombardment protocol has been employed in our laboratory to deliver hundreds of different DNA
constructs into C. elegans and generally produces 10-20 independent transformed lines per bombardment. Roughly half of these lines are integrated, and
more than half of the integrated lines are typically viable as homozygotes. We generally perform 3-4 bombardments on one day
of each week, although this throughput depends upon continual passaging and cleaning of unc-119 mutant stocks. Roughly 3 ml of packed adult hermaphrodites are required for one successful bombardment, so large scale growth
of unc-119 mutants is the most time-consuming and laborious step in the protocol.

4.1. Making egg media

Autoclave the metal carafe of a Waring blender, or sterilize a glass blender carafe by running with 10% bleach, followed by
rinsing with sterile water. Spray the outer shell of raw chicken eggs obtained from the grocery store with 70% ethanol, and
wipe away the liquid using Kimwipes™. Crack the eggs and collect their contents in a sterile beaker. Discard the shells. Boil
25 ml of water per egg in an Erlenmeyer flask in a microwave oven. Pour the contents of the eggs into the blender, add the
boiling water, and run the blender. When the solution becomes homogenous, dump the contents of the blender into a sterile
beaker, and let the froth settle for one hour. Pipette the egg solution into 6 ml aliquots in 15 ml conical tubes, and incubate
the tubes at 65°C for 1 hour in a water bath to inactive endogenous lysozyme. Allow the tubes to cool to room temperature,
and freeze them for long-term storage at −80°C.

4.2. Preparing concentrated bacteria

A concentrated HB101E. coli stock for egg plates is prepared by seeding 250 ml of 2xYT medium, in a 2 L flask, with a single HB101 colony. Shake the flask overnight (37°C at 225 rpm). The following day, aliquot the culture into 50 ml conical tubes. Before
use, pellet the bacteria at 4000 rpm for 15 minutes. Discard the supernatant. Resuspend the bacterial pellet by vortex in
a volume of M9 buffer roughly equal to the volume of the pellet. Store the unused aliquots of bacteria at 4°C. HB101 cultures used for seeding smaller growth plates, prior to the egg plate stage, are not concentrated.

Prepare starter cultures of DP38unc-119(ed3) worms on 6 cm NGM plates that have been seeded with an HB101 lawn. HB101 is preferred in this protocol over the typical OP50 strain of E. coli used for C. elegans genetics, because HB101 produces a thicker lawn (more food for the animals), and because HB101 is an easier strain for the mutant animals to ingest (see C. elegans feeding). Some protocols for large scale growth of C. elegans call for the use of growth plates highly enriched in peptone, but we have found that unc-119 mutants grow best on standard NGM or NGM-lite (Maintenance of C. elegans). unc-119 mutants also grow better at low temperature (15-20°C) than high temperature (25°C). Allow the worms to starve out the plates.
Divide one complete, recently starved, 6 cm plate to four new 10 cm HB101 seeded NGM/NGM-lite plates. Spread small chunks containing unc-119 worms to several locations on the large HB101 lawn to facilitate even distribution of the worms as they grow, since unc-119 worms are nearly paralyzed and never stray very far from the area in which they hatch. For the same reason take care to place
the chunks face down, with starved worms in direct contact with the bacterial lawn. Allow the 10 cm plates to exhaust their
food supply, typically nine to eleven days at 20°C. Starved worm cultures can be saved at 15-20°C for up to 2 weeks prior
to their use in inoculating egg plate cultures. Discard any plates bearing visible contamination before starting egg-plate
cultures.

Note: The growth of starter cultures of unc-119 mutant worms is quite time consuming, generally taking 2-3 weeks for completion. If bombardments are to be performed fairly
regularly, then 6 cm and 10 cm NGM/HB101 cultures of unc-119 mutant worms can be started by chunking on a weekly basis. This approach requires a relatively small effort, and creates
a situation in which there are always worms available to use to start egg plate cultures, reducing the required lead-time
for bombardment to 4 days. Because of their paralysis and slow growth, unc-119 mutant cultures are more prone than wild-type cultures to accumulate contaminating mold and bacteria, and must be cleaned
by basic hypochlorite digestion when contamination occurs (Maintenance of C. elegans).

4.4. Starting egg plate cultures

Four 15 cm egg plate cultures are required to obtain enough animals for one bombardment. One 10 cm culture is used to seed
one 15 cm egg plate culture. For one bombardment, wash worms from four recently starved 10 cm unc-119 worm cultures. Wash the plates twice, using 5 ml of 100 μg/ml streptomycin-spiked M9 salts per 10 cm plate, collecting the
liquid and worms in one 50 ml conical tube. Next, pellet the worms by centrifugation for 2 min. at 1500 rpm. Discard the supernatant.

Spin down one 50 ml tube of HB101 bacteria per set of four egg plates at 3000 rpm for 5 min. Discard the supernatant. Add 500 μl of the streptomycin-spiked
M9 buffer to the pellet and resuspend the bacteria by brief vortexing. Mix the resuspended bacteria with one thawed 6 ml aliquot
of egg medium, then mix it with the worm pellet obtained from four starved 10 cm culture plates. Aliquot 25% of the worm/HB101/egg mixture onto the surface of each of four 15 cm NGM lite plates. Use a disposable bacterial spreader to evenly distribute
the worm/HB101/egg mixture on the plate (Figure 1A). It is necessary to mix the worms with the bacterial media prior to spreading because the nearly paralyzed unc-119 strain cannot evenly self-distribute. Grow the unc-119 egg plate cultures for approximately 4 days at 20°C, until most animals are gravid adults, before using the worms for bombardment.
The time to harvest can vary and must be determined by inspection of the cultures (see Figure 1B).

Figure 1. Growth of unc-119(ed3) worms for bombardment. (A) Spreading mixture of worms/egg/HB101 across surface of 15 cm NGM-lite plate. (B) Plate ready to harvest for bombardment. After spreading, grown for 4 days at
20°C. Note that worms are found mostly in large piles with remaining food. The majority of the animals are gravid adults.
(C) Vortexing gold particles at low speed using foam tube holder insert. A vortexer with such an insert is required equipment
for the long vortexing steps associated with precipitation of DNA onto the gold particles.

4.4.1. Alternatives:

Large scale unc-119 worm cultures can be grown in liquid (S medium, see Maintenance of C. elegans), or on a very large number of more standard 10 cm HB101 seeded growth plates (without egg media, see Transgenic solutions for the germline). Liquid cultures typically require a shaking incubator equipped with refrigeration to maintain growth temperatures ≤ 20°C.
In our experience the egg plate method requires the least effort to produce the large number of animals required for successful
bombardment.

4.5. Preparing gold particles

Use a siliconized microcentrifuge tube to prepare gold particles. Put the siliconized microcentrifuge tube on a balance, and
zero the balance. Weigh out 30 mg of gold particles in the tube. Add 1 ml of 70% ethanol. Vortex the tube for 5 min., using
a foam insert in the vortexer to hold the tubes (Figure 1C). Stop the vortexer and allow the tube to rest for 15 min. Spin the tube briefly and discard the supernatant. Wash the particles
three times with 1 ml of sterile water by adding the water, vortexing, and then tap spinning the tube (i.e., spin very briefly
using the “pulse” button on the centrifuge). When removing the wash water, touch the pipette tip to the side of the tube opposite
the gold. After removing the water from the third wash, resuspend the gold in 0.5 ml 50% sterile glycerol. The final gold
concentration is 60 mg/ml. The gold may be stored for at least 1-2 months at 4°C. 50% glycerol stocks should be stored in
the dark.

4.6. Drying 10 cm plates

Worms grown on four egg plates are harvested in buffer and placed on one seeded 10 cm NGM/NGM-lite plate. This process requires
that the receiving NGM plate be dry enough to soak up the transferred buffer, such that one ends up with very tight packing
of the worms to be bombarded. In order to ensure that the 10 cm NGM plates to be bombarded will not be too wet, they must
be dried prior to application of worms in a 37°C incubator, with the lid slightly ajar. Drying generally takes 2-4 hours.
Afterwards, place the plates on Saran™ wrap covered ice to chill (Figure 2A). Chilling the plates keeps the worms from moving prior to bombardment, keeping them in a tightly packed monolayer, or even
bilayer, covering the entire plate surface. The Saran wrap helps prevent contamination of the worms by the ice, which tends
to be a fruitful source of unwanted bacteria and mold (Figure 2A).

Figure 2. Worms ready to bombard. (A) Approximately 3 ml of packed worms coating the surface of a chilled 10 cm NGM-lite plate (HB101 seeded). A confluent layer of worms completely coats the surface of the plate. Chilling the plate stops worm movement that
creates gaps in the coverage. Plastic wrap covering the ice reduces contamination of the worms with bacterial and fungal microbes.
(B) Confluent worm plate in bombardment chamber. The plate is attached to the shelf using at least five pieces of rolled
up lab tape. Note position relative to pressure divider and macrocarrier holder components of Hepta adapter.

4.7. DNA precipitation onto gold particles

Check stock of 2.5 M CaCl2 for undissolved precipitate. If the solution contains undissolved precipitate, warm it in a 37°C water bath until the precipitate
re-enters solution. Thaw a 0.1 M spermidine aliquot from −80°C stock. Mix 8-16 μg of plasmid DNA, in a total volume of 50
μl, with 100 μl of well-suspended gold particle solution in 50% glycerol, using a siliconized microfuge tube. If an unc-119(+) gene is already present in the plasmid to be used in bombardment, and the total size of the plasmid is less than 10kb, then
10 μg of DNA is a good amount to use. For larger plasmids, a proportionately greater amount of DNA is often required to obtain
good results. Also, if the unc-119(+) gene is on a separate plasmid from the one you wish to bombard, using a mixture of two plasmids (8-10 μg of each) is best.
Co-bombardment of two plasmids works, but only about two-thirds of the integrated lines contain the unselected plasmid. Integration
of a rescuing unc-119 gene within the plasmid of interest improves the efficiency of useful transgene recovery.

After combining the gold particle solution and DNA, vortex the tube for at least 1 minute (Figure 1C). Next, add 150 μl 2.5M CaCl2. Vortex the solution again, for at least 1 minute. Then, add 60 μl 0.1M spermidine. Vortex the tube 3 to 5 min. (Figure 1C). Re-freeze remaining spermidine stock (store frozen at −80°C). Tap spin the solution. Pulse to 6,000 rpm then release. Spinning the solution too long or at much higher g-force will make resuspension difficult or impossible. After the tap spin, remove the supernatant. Add 300 μl of 70% ethanol. Vortex the solution very well. The precipitated DNA
makes the gold very sticky. Tap spin the solution again. Remove the supernatant. Then, add 170 μl of 100% ethanol, and vortex
the tube on high for 5 to 10 min. Then, turn down the vortex to a low speed until the DNA-coated gold is actually used (Figure 1C). All vortexing steps are typically performed using a foam insert in the vortexer that can hold the tubes without supervision
(Figure 1C). We find that a rubber mouse pad placed under the vortexer can prevent its migration across the bench during the long mixing
steps.

Note: The DNA precipitation protocol is based upon a DNA starting volume of 50 μl. If using a larger starting volume increase CaCl2, spermidine, and ethanol (but not gold particle) volumes proportionately.

4.8. Preparing the macrocarriers and PDS-1000/Hepta apparatus

Add some dryerite covered by a Kimwipe to an empty 15 cm petri dish. With forceps, dip seven macrocarriers per bombardment,
one at a time, into 100% ethanol and lay them on the kimwipe in the plate to dry (Figure 3A). Keep the lid slightly ajar in order to let the macrocarriers dry.

Figure 3. Preparing Macrocarriers. (A) Drying macrocarriers after dipping in ethanol. (B) Pipetting DNA-coated gold particles onto macrocarriers. (C) Drying
DNA-coated gold particles on macrocarriers. Note that gold particles are centered over holes in macrocarrier holder. (D)
Pressing tool used to firmly seat macrocarriers in macrocarrier holders. Apply pressure while using a twisting motion to
firmly seat the macrocarriers such that they remain in place when carrier is inverted.

Clean the bombardment chamber with ethanol and Kimwipes (Figure 4A). Wash the target plate shelf in a similar manner (Figures 2B and 4A). Roll up four or five pieces if lab tape, with the sticky part on the outside, in order to tape the worm plate to the shelf.
Place the shelf in the bottom slot of the bombardment chamber (Figure 2B). Wipe down the metal parts of the macrocarrier holders with ethanol and Kimwipes (Figure 3B). Load the macrocarriers into the lid of the macrocarrier holder with forceps. Wipe the red plastic pressing tool with ethanol
and Kimwipes (Figures 3D and 4A). Press the macrocarriers into the lid with the red pressing tool using a twisting motion. Add 20 μl of DNA-coated gold particles
to the center of each macrocarrier, try to cover only the central area of the macrocarriers which lie above the holes in the
metal macrocarrier holder (Figure 3B, C). Then split up any remaining DNA-coated gold particles among the macrocarriers, again only covering the central areas (Figure 3B, C). Let the gold solution on the macrocarriers dry (Figure 3C). A fume hood can speed up the drying process.

4.9. Preparing the worms

Wash unc-119(ed3) mutant worms from 4 egg plates with sterile streptomycin-spiked M9 salts (Figure 1B). It is easiest to cover the worms with M9 buffer and allow them soak for 10 min. or more. Disposable scrapers can be used
to dislodge worms. Remove the worms in M9 buffer from the plates with a pipette. Transfer the worms to a 50 ml conical centrifuge
tube. Centrifuge the worms for 2 min. at 1500 rpm in a table-top centrifuge. Pipette off the supernatant, and discard. Resuspend
the worms in M9 in a total of 25 ml. Allow the adult worms to settle to the bottom of the tube by gravity for 15-30 min. Then,
remove the supernatant. The purpose of letting the worms settle by gravity is to enrich for adults over larvae, and to remove
egg debris. Pipet the adult worm pellet derived from four 15 cm egg plates onto one dried, ice-chilled, 10 cm HB101 plate (Figure 2A). Draw up the worms from the bottom of the pellet. 2.75 ml of packed worm pellet is the perfect amount for one 10 cm target
plate. Let the plate rest on Saran wrap covered ice for at least 30 min. to allow the plates to absorb the liquid. A confluent layer
of worms should coat the whole plate, and the worms should not be floating in liquid.

4.10. Test firing the apparatus

Before an actual bombardment, test firing the apparatus is recommended. To test fire the apparatus, dip a rupture disk in
100% ethanol with forceps and shake off the excess ethanol. Place the rupture disk into the top of the pressure divider (Figure 4B). Screw the pressure divider into the bombardment chamber and tighten using the small torque wrench provided with PDS-1000
(Figure 4C). Close the door of the bombardment chamber. Open the helium and adjust the pressure to at least 200 psi higher than the
amount needed to rupture the disk. We usually use a 1350 psi rupture disk, thus requiring at least 1550 psi in pressure from
the helium tank. Turn on the vacuum pump. Turn on power to the bombardment chamber (Figure 4A, switch 1). Place the vacuum switch to the vacuum position (Figure 4A, switch 2), and hold it until the needle on the vacuum gauge reaches 28 ”Hg (Figure 4A). Then, very quickly turn the vacuum switch to hold, skipping the vent level of the switch. Press and hold down the fire
button, allowing gas pressure to slowly build behind the rupture disk (Figure 4A, switch 3). Gas pressure at the level of the rupture disk is monitored via the helium pressure gauge on top of the PDS-1000
(Figure 4A). Release the fire button once the rupture disk breaks. One should hear a distinct pop. One should also see that the helium
pressure gauge on top of the PDS-1000 approached 1350-1500 psi, then suddenly dropped to 0 psi. Turn the vacuum switch to
vent, and turn off power to the vacuum pump and PDS-1000 (Figure 4A, D). Unscrew the pressure divider using the torque wrench (Figure 4C). Remove the burst rupture disk using forceps and discard.

4.11. Bombardment

Wipe down the two parts of the pressure divider with ethanol and Kimwipes. Dip a rupture disk into ethanol with forceps and
shake off the excess ethanol. Place the rupture disk into the pressure divider (Figure 4B). Screw the pressure divider into the nozzle of the bombardment chamber using the torque wrench (Figure 4C). Place an autoclaved stopping screen onto the pegs of the lower half of the macrocarrier holder. Invert the top half of
the macrocarrier holder (containing the macrocarriers), and place it onto the pegs of the lower half of the macrocarrier that
lies beneath the stopping screen. Place the assembled macrocarrier apparatus onto the metal shelf, and rotate the lever to
lock the shelf onto the macrocarrier apparatus. Insert the macrocarrier apparatus shelf complex into the bombardment chamber
using the second shelf slot from the top, directly below the Hepta pressure divider (Figure 2B). Rotate the lever of the macrocarrier apparatus such that the holes of the complex align with the holes of the pressure
divider. Tape down the worm plate for bombardment to the target plate shelf (Figure 2B). If tape is not used, the plate will jump during rupture of the disk, interfering with successful bombardment.

Close the door of the bombardment chamber. Open the helium, and adjust the pressure to at least 200 psi higher than the amount
needed to burst the rupture disk (e.g., 1550 psi). Turn on the vacuum pump (Figure 4D). Turn on power to the bombardment chamber (Fig 4A, switch 1). Place the vacuum switch to the vacuum position (Figure 4A, switch 2), and hold it until the needle on the vacuum gauge reaches 28 ”Hg (Figure 4A). Then, very quickly turn the vacuum switch to hold, skipping the vent level of the switch. Press and hold down the fire
button, allowing gas pressure to slowly build behind the rupture disk (Figure 4A, switch 3). Gas pressure at the level of the rupture disk is monitored via the helium pressure gauge on top of the PDS-1000
(Figure 4A). Release the fire button once the rupture disk breaks. One should hear a distinct pop. One should also see that the helium
pressure gauge on top of the PDS-1000 approached 1350-1500 psi, then suddenly dropped to 0 psi. Turn the vacuum switch to
vent, and turn off power to the vacuum pump and PDS-1000 (Figure 4A, D). Remove the worm plate and set aside at room temperature for at least 30 min. Wash the worms off of the plate with M9/strep,
and distribute them equally among twenty 10 cm, HB101-seeded, NGM-lite plates.

While worms are resting, close the helium gas tank and release the remaining pressure in the pressure regulator that is attached
to the helium tank. Unscrew the pressure divider using the torque wrench (Figure 4C). Remove the burst rupture disk using forceps and discard. Save the used stopping screen. Used stopping screens may be rinsed
and autoclaved for re-use. Wipe down every part of the machine with ethanol and Kimwipes. When finished with all bombardments,
jiggle both the vent and fire switches to release any residual helium pressure in the apparatus. This is important. Residual
pressure can damage the machine!

4.12. Recovering transgenic lines

Grow the bombarded worms, which are now on twenty-one 10 cm seeded plates (the original plate is saved) at 25°C for 10-14
days or more. Non-Unc transgenic animals can be recovered by simple visual screening, looking for fast moving animals after
tapping the plate on the bench or microscope stage. In many cases dozens or hundreds of moving animals are visible. Alternatively
recover non-Unc worms by “reverse chunking”, which is performed by placing an agar chunk from a seeded plate, bacterial side
up, in the center of the starved bombardment plate. Animals with normal movement will seek out the food on the top of the
agar chunk and are easily retrieved the day after placing the bait (Kevin O'Connell, personal communication, see Reverse chunking, a simple and effective method for identifying unc-119 transgenics). Single out non-Uncs from each plate to screen for integrated lines. We generally single out 2 worms per 10 cm plate. We
assume that all non-Unc animals from the same founder plate are likely to be siblings carrying the same transgene, although
in some cases clear differences in expression level or pattern in animals from the same plate suggest that more than one independent
line may be present. If possible we choose larval animals as founders, since adults from such plates are often of a post-reproductive
age. Those plates that produce all non-Unc progeny are homozygous integrants. Plates that segregate less than 100% non-Uncs
are either heterozygous integrants or extrachromosomal array lines. Heterozygous integrants should segregate about three-fourths
non-Uncs and one-fourth Uncs, while arrays vary widely in their maintenance through meiosis with some array lines maintained
at around this level. To identify integrated lines initially isolated as heterozygotes, we single out an additional nine non-Unc
worms from candidate plates, again assaying in the next generation for 100% transmission of the transgene. We often screen
lines for desired expression properties prior to such additional singling out efforts.

Figure 5. Bombardment Flowchart.

5. Acknowledgements

We thank Geraldine Seydoux, Shai Shaham, and Ron Plasterk for providing bombardment protocols developed in their laboratories
that proved very useful in developing this protocol. We thank Kevin Huang for help with the figures. We also owe thanks to
Ken Sato and Miyuki Sato for working out the initial successful version of this protocol in the Grant lab. We thank Richard
Davis for suggesting the use of custom made two micron gold particles as DNA carriers for bombardment. This work was supported
by NIH grants GM067237 and GM103995 to B.D.G.